Despite the availability of highly active antiretroviral therapy (HAART or ART), opportunistic infections (OIs) remain the leading cause of considerable morbidity and mortality in HIV infected persons as stated by the CDC in MMWR, April 10, 2009. This is particularly important with the leading OI, pneumonia (PCP), caused by Pneumocystis jirovecii. Trimethoprim/sulfamethoxazole (TMP/SMX), the combination of a dihydrofolate reductase (DHFR) inhibitor (TMP) and a dihydropteroate synthase (DHPS) inhibitor (SMX) for PCP is the first- line agent. The failure of this option due to adverse reactios and resistance to the sulfa drug as well as TMP along with the failure rate and adverse reactions of second-line agents necessitates the urgent need for alternate agents. Our group has isolated and characterized the elusive DHFR from the human pathogen Pneumocystis jirovecii (pjDHFR) and shown it to be distinct and different from its surrogate Pneumocystis carinii (pcDHFR) (occurs in rodents), with respect to inhibitory activities of therapeutic agents. n addition, we have identified two series of compounds (1, 3, 5 and 6) with selectivity for pjDHFR over hDHFR of 19-99-fold with picomolar and nanomolar Ki and IC50 values for pjDHFR. To our knowledge, we are the only group with access to pjDHFR and compounds that have both high selectivity (19-99-fold) and potency (picomolar and nanomolar) against pjDHFR (compared to hDHFR). Recently we have also cloned and expressed clinically relevant, TMP-resistant double mutants of pjDHFR and found that our compounds retain nanomolar inhibition against the double mutant pjDHFR in the face of TMP's 500-fold resistance, and will also use these in our evaluations. This is a paradigm changing event in the evaluation of drugs for P. jirovecii infection that up until now has utilized the surrogate P. carinii for DHFR as well as in vivo models. The Specific Aims are to: 1) synthesize proposed compounds in Series I-XII; 2) evaluate the compounds as inhibitors of hDHFR and wild type and resistant mutant pjDHFR; 3) evaluate selected analogs from Aim 2 in human embryonic lung fibroblasts for toxicity; 4) evaluate selected analogs (5) from Aims 2 and 3 for serum binding, metabolic stability and pharmacokinetics and develop and evaluate in a mouse model of P. jirovecii infection. X-ray crystal structure determination of 1, 3 and 5 and selected compounds from Series I-XII with pjDHFR and hDHFR will be done by Dr. Cody to afford a molecular understanding of the selectivity and potency of the analogs. This study will determine the structural requirements for potent and selective inhibition of pjDHFR and will assist in future drug design and pharmacophore generation. In addition it will provide, for the first time, the evaluation of compounds in a P. jirovecii animal model based on DHFR from the human pathogen (P. jirovecii) rather than a surrogate (P. carinii). The study should identify potential compounds for clinical evaluation against PCP and resistant PCP to be used alone or in combination.